Characterisation of the structure of ocr, the gene 0.3 protein of bacteriophage T7

The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes. We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr. Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 ± 0.7:1 and mass of 27 kDa. The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially. Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers. Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94. Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better. These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface. This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes.     

Structure, chromosomal assignment, and expression of the gene for proteinase-3. The Wegener's granulomatosis autoantigen.

Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It degrades a variety of extracellular matrix proteins including elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters. It is identical to the target autoantigen (c-ANCA) associated with Wegener's granulomatosis and to myeloblastin, a serine proteinase first identified in HL-60 leukemia cells. In this study, the gene encoding PR-3 was cloned and sequenced. The gene spans approximately 6.5 kilobase pairs and consists of five exons and four introns. The genomic organization of PR-3 is similar to that of the other serine proteinases expressed in hemopoietic cells. Each residue of the catalytic triad of PR-3 is located on a separate exon, and the positions of the residues within the exons are similar to those in human leukocyte elastase and cathepsin G. The phase and placement of the introns in the PR-3 gene are also similar to those in human leukocyte elastase and cathepsin G. The 400-base pair (bp) 5'-flanking sequence of the PR-3 gene contains a TATA box at position 379. There is no CAAT box promoter element. The 3'-untranslated region is 200 bp, extending from a TGA stop codon to the site of polyadenylation 10 bp after the canonical AATAAA signal. Amplification of PR-3 from a human/hamster hybrid cell line localizes the gene to human chromosome 19. Evidence from Northern analysis suggests that PR-3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development.     

Renal capillariasis in the small Indian mongoose, Herpestes auropunctatus.

A Capillaria sp. was recovered from the kidneys of 28 (93.3%) of 30 small Indian mongooses (Herpestes auropunctus) collected in St. Lucia, West Indies. The nematodes were embedded within distended pelvic fornices of the kidney and surrounded by accumulations of eggs. A chronic, low-level inflammation of the transitional epithelium was characterized by hyperplasia, giant cells surrounding embedded eggs and a plasmacytic infiltration. This is the first record of a capillarid nematode from the kidney of the mongoose.     

The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function

Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defence and a wide range of developmental processes in fungi, plants, and animals. Despite their dramatic diversification in organisms, TLPs appear to have originated in early eukaryotes and share a well-defined TLP domain. Nonetheless, determination of the roles of individual members of the TLP superfamily remains largely undone. This review summarizes recent advances made in elucidating the varied TLP activities related to host resistance to pathogens and other physiological processes. Also discussed is the current state of knowledge on the origins and types of TLPs, regulation of gene expression, and potential biotechnological applications for TLPs.     

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis

Introduction

The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA).

Methods

CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests.

Results

Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor α, interferon γ and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help.

Conclusions

The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.     

A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) assays are commonly used for measuring enzymatic activity in clinical and biological samples. The fluorimetric procedure described is sensitive, rapid and involves unsophisticated procedures and inexpensive reagents. It uses the substrate hippuryl-L-histidyl-L-leucine, and the fluorescent adduct of the enzyme-catalyzed product L-histidyl-L-leucine is quantified fluorimetrically. This assay has been adapted for a 96-well plate format that produces comparable data to previously described assays and has the advantage of greater efficiency with respect to both time and reagents. The protocol can be used for routine purposes or for more detailed kinetic analyses. The apparent Km and kcat values for purified testis ACE determined from a double reciprocal plot were 3.0 mM and 195.7 s(-1), respectively. The protocol can be completed within 4 h.     

Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47(phox-/-) than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox-/- compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 +/- 2.4 and 9.2 +/- 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation.